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Abstract:

The present invention relates to new plasma or new platelet-rich plasma
preparations, new cell dissociation methods, new cell associations or
compositions, a method of preparation thereof, a use thereof, devices for
the preparation thereof and preparations containing such a platelet-rich
plasma preparation and cell associations or compositions. Specifically,
the invention provides plasma or platelet-rich plasma alone or in cell
combinasons preparations for use in tissue regeneration and bone
regeneration and pain reduction.

Claims:

1. An isolated cell composition comprising: a) plasma; b) platelets at a
concentration of at least 300.times.10.sup.9 cells/L; c) white blood
cells at a concentration of at least 7.0.times.10.sup.9 cells/L; and d)
fibrinogen at a concentration of at least 3 mg/L; and wherein the
erythrocyte concentration is less than 0.6.times.10.sup.12 cells/L.

2. An implantable device for use in tissue regeneration therapy
comprising: a) a permeable core comprising a cell composition comprising:
i) plasma; ii) platelets at a concentration of at least
300.times.10.sup.9 cells/L; iii) white blood cells at a concentration of
at least 7.0.times.10.sup.9 cells/L; iv) fibrinogen at a concentration of
at least 3 mg/L; and v) a cell extract where cells are selected from
dermal cells, keratinocytes, fibroblasts, melanocytes and Langheran's
cells; fat cells; bone marrow cells; muscle cells; osteoblasts;
chondrocytes; periosteal membrane cells; corneal cells; umbilical cord
cells; Schwann cells, tendon cells and pancreas islet cells, wherein
cells are at a concentration of about 10.sup.5 to about 10.sup.6 cells/L
and wherein the erythrocyte concentration of said cell composition is
less than 0.6.times.10.sup.12 cells/L; and b) an external jacket
surrounding said core, said jacket comprising a biocompatible material or
a bioresorbable material.

3. A device for the preparation of a platelet concentrate from whole
blood comprising a separator tube wherein the separator tube is selected
from: a glass separator tube containing a polyester-based thixotropic gel
and a buffered sodium citrate solution at 0.10 M; and a polyethylene
terephthalate separator tube containing a highly thixotropic gel formed
by a polymer mixture and an anhydrous sodium citrate at 3.5 mg/mL;
characterised in that the device has an inlet for introducing said whole
blood, is held in a vacuum intended to aspirate the whole blood sample,
is sterile, has a usable vacuum of or about 8 to about 10 mL and is
suitable for undergoing centrifugation.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Ser. No. 12/438,236,
filed Feb. 20, 2009, which is the U.S. national stage application of
International Patent Application No. PCT/EP2007/058695, filed Aug. 21,
2007, the disclosures of which are hereby incorporated by reference in
their entirety, including all figures, tables and amino acid or nucleic
acid sequences.

FIELD OF THE INVENTION

[0002] The present invention is related to the field of tissue
regeneration, especially skin, cartilage, muscle, tendon, adipose tissue,
cornea, peripheral nerves, spine and bone regeneration. It concerns more
particularly new cell preparations as a biological scaffold, a method of
preparation thereof, a use thereof, a device for the preparation thereof
and preparations containing such cell preparation for extemporaneous use.

BACKGROUND OF THE INVENTION

[0003] The importance of biological autologous materials in the healing
process has been well documented. Most importantly, two biological
autologous materials have been shown to be directly implicated in the
formation of the structure of blood clots, which provide a haemostatic
barrier whose role is to ensure hemostasis and seal the wound: (1)
fibrin, which derives from the separation of plasma fibrinogen into two
strands through the action of thrombin, and (2) the activated membranes
of platelets.

[0004] The wound healing process is generally presented as the succession
of a coagulation phase, an inflammatory process and a regeneration
process.

[0005] The coagulation phase (blood clotting or clot formation) is a
complex process whereby a damaged blood vessel wall is covered by a
fibrin clot to stop hemorrhage and the repair of the damaged vessel is
initiated by the release in large quantities of cytokines and growth
factors from platelet alpha granules. The formation of blood clots
(formed in physiological conditions by fibrin, platelets and red blood
cells, among other blood components) is a natural phenomenon that results
from tissue trauma and its role in the wound healing process, as well as
in the union of bone fractures, is well-known.

[0006] The inflammation process, which follows the formation of a blood
clot, is stimulated by numerous vasoactive mediators and chemotactic
factors (specific signals in the form of proteins) released by white
blood cells and platelets. These signals attract macrophages that "clean"
the site from bacteria and foreign particles as well as red blood cells
before the migration of new cells.

[0007] The tissue regeneration phase involves the chemoattraction and the
mitosis of the undifferentiated cells in the scaffold (or growth matrix)
formed by the blood clot. The new cells which multiply under the
stimulation of platelet growth factors will replace damaged or destroyed
cells injured by macrophages.

[0008] Growth factors and numerous plasma proteins, also called signaling
molecules, which promote cell migration and division within blood clots,
play a crucial role in the wound healing process.

[0009] Theoretically, it is possible to amplify the effects of these first
phases in the wound-healing cascade by discarding the red blood cells and
increasing the concentration of growth factors.

[0010] Blood clotting amplification can be defined as the formation of an
"enriched clot (EC)". ECs are obtained through the use of platelet
concentrates and have been described in Platelets and Megacaryocytes
2004, vol 1 & 2, as "Structure and signals", Ed. Gibbins and
Mahaut-Smith, Humana Press, New Jersey.

[0011] Platelet-rich plasma (PRP) can be defined as an autologous
concentrate of platelets in a small volume of plasma; it has been
developed as an autologous biomaterial and has proven to be useful in the
healing and regeneration of tissues (Marx et al., 2004, J. Oral
Maxillofac. Surg., 62, 489-496). PRP not only consists in a platelet
concentrate but also contains growth factors (such as platelet-derived
growth factor: PDGF, vascular endothelial growth factor: VEGF,
transforming growth factor: TGF and epidermal growth factor: EGF) that
are actively secreted by platelets and are known to have a fundamental
role in wound healing initiation process.

[0012] For example, PDGF is known to initiate connective tissue healing,
including bone regeneration and repair. PDGF also increases mitogenesis
(healing cells), angiogenesis (endothelial mitosis into functioning
capillaries) and macrophage activation. VEGF released by the leukocytes
is also known to have potent angiogenic, mitogenic and vascular
permeability-enhancing activities on endothelial cells. TGF-β
promotes cell mitosis and differentiation for connective tissue and bone,
acts on mesenchymal stem cells, preosteoblasts and fibroblasts and
inhibits osteoclast formation. EGF is known to induce epithelial
development and promote angiogenesis.

[0013] Platelet concentrates are generally used in dental implantology and
bone surgery, notably in the USA. Various techniques of preparation of
PRP by centrifugation processes have been developed. However, due to the
sensitivity of the platelet cells and the variability of the efficiency
of the methods of separation of the platelets from the red blood cells, a
great variability exist among the methods used for the preparation of
platelet concentrates (Marx et al., 2004, above; Roukis et al., Adv.
Ther., 2006, 23(2):218-37): for example, the laboratory is material for
in vitro diagnostic which is used for platelet preparation, leads to a
poor platelet and other plasma components yield (Marx et al., 2004,
above: Anitua 35%, Landsberg 30%, Clinaseal 39%, ACE surgical 33%,
Curasan 29%). The automated settings from Biomet PCCS & GPS (Marx et al.,
2004, above), which not only present the drawback of being a complex
process with prohibitive costs for the process of a blood sample, lead to
only a yield of 61% and SmatPreP from Harvest Technology 62%. In those
systems, there is obviously an important loss of valuable biologic tissue
from the patients, therefore there is the need for the development of a
reliable process collecting the plasma cells with high yields, easy to
use and cost effective.

[0014] It has been recently demonstrated that the positive effects of
platelet-rich plasma on bone regeneration spans a limited range of
platelet concentration and revealed that an inhibitory effect occurs in
the presence of more than 106 platelets per μl, which is 3 to 4
times baseline counts (Weibrich et al., 2004, Bone, 34(4):665-71).

[0015] In addition, the obtaining of platelet concentrates still needs the
use of relatively complex kits and costly dedicated machinery and the
equally costly involvement of specialized technicians. This drawback
makes the current known methods of preparation of PRP not adapted to a
point-of-care use.

[0016] Further, the preparation of cells in view of cellular or tissue
regeneration for use in transplantation, post-operative regeneration or
for aesthetic purpose is faced to the long-term conservation problem of
cells and tissues. Tissue or cell cryoconservation is generally used for
the long-term maintaining of tissues or cells, notably platelets, but
this technique has shown serious drawbacks and problems such as crystal
formation, osmotic problems, aggregation, inhibition of protein synthesis
ability, stress protein expression in response to thermal stress, etc.
Therefore, tissue or cell cryoconservation is known to alter the cell
viability and stability (Agence francaise de securite sanitaire, 2003;
Arnaud et al., 1999, Cryobiology, 38, 192-199; Tablin et al., 2001,
Cryobiology, 43(2), 114-23). Some of the cryoconservation side effects
may be limited by the use of anti-freezing agents such as DMSO or
glycerol or other cryopreservatives (U.S. Pat. No. 5,5891,617, Oh et al.,
Cornea, 26, 840-846) but the concentration of these agents has to be
adapted to limit their toxicity and side effects.

[0017] Therefore, there is a need for new or alternative method of
preparation of cells and tissues suited for use extemporaneously while
preserving their integrity, notably in terms of growth factors secretion
ability and viability.

SUMMARY OF THE INVENTION

[0018] The invention relates to new cell preparation, a method of
preparation of new cell preparations, a use of such cell preparations
containing such as platelet cell preparations, optionally admixed with a
cell extract, such as an autologous extract of keratinocytes, bone marrow
cells, fibroblasts, periosteum or corneal cells, melanocytes and
Langerhans cell; fat cells; muscle cells such as myoblasts and satellite
cells; osteoblasts; chondrocytes; umbilical cord cells; Schwann cells or
Achilles tendon cells.

[0019] The process for the preparation of a platelet concentrate
composition according to the invention constitutes a reliable process
collecting 95%+/-5 of the plasma cells, easy to use and cost effective
(Borzini P. et al., in preparation).

[0020] In a first aspect, the present invention provides a process for the
preparation of a platelet concentrate composition, comprising the steps
of:

[0021] a) Centrifuging whole blood in a separator tube selected from:

[0022] a glass separator tube containing a polyester-based thixotropic
gel and a buffered sodium citrate solution at 0.10 M; and

[0023] a
polyethylene terephthalate separator tube containing a highly thixotropic
gel formed by a polymer mixture and an anhydrous sodium citrate at 3.5
mg/mL;

[0024] b) Separating the enriched platelet rich plasma from the
full plasma by removing half of the supernatant containing the platelet
poor plasma;

[0025] c) Re-suspending the enriched plasma; wherein the
centrifugation step is performed at a force of or about 1500 g up to
about 2000 g in a sufficient length of time to form a barrier between the
plasma containing the platelets, the lymphocytes and the monocytes and
the pellet containing the erythrocytes; the separation step b) is made by
collecting the supernatant from atop of said barrier and wherein the
enriched plasma is enriched in leucocytes, thrombocytes and adhesion
proteins (for example, fibronectin) as compared to native whole blood.

[0026] In a second aspect, the present invention provides an isolated
platelet concentrate is composition comprising:

a) plasma; b) platelets at a concentration of at least 300×109
cells/L; c) white blood cells at a concentration of at least
7.0×109 cells/L; d) fibrinogen at a concentration of at least
3 mg/L; and wherein the erythrocyte concentration is less than
0.6×1012 cells/L.

[0027] In a third aspect, the present invention provides a wound healant
composition comprising:

a) plasma; b) platelets at a concentration of at least 300×109
cells/L; c) white blood cells at a concentration of at least
7.0×109 cells/L; d) fibrinogen at a concentration of at least
3 mg/L; e) coagulation activator in a vol. ratio (platelet
concentrate:coagulation activator) of about 10:1 to about 10:3; f)
optionally an autologous cell extract, such as an extract of
keratinocytes, bone marrow cells, osteoblasts; chondrocytes, fibroblasts,
periosteum or corneal cells, melanocytes and Langerhans cell; fat cells;
muscle cells such as myoblasts and satellite cells; umbilical cord cells;
Schwann cells, tendon cells or pancreas islet cells; and wherein the
erythrocyte concentration is less than 0.6×1012 cells/L.

[0028] In a fourth aspect, the present invention provides a process for
the preparation of a wound healant composition comprising:

[0029] a)
Providing a platelet concentrate of the invention;

[0030] b) Admixing the
platelet concentrate with a coagulation activator in a vol. ratio
(platelet concentrate:coagulation activator) of about 10:1 up to about
10:3;

[0032] In a fifth aspect, the present invention provides a device for the
preparation of a platelet concentrate from whole blood comprising a
separator tube wherein the separator tube is selected from:

[0033] a glass separator tube containing a polyester-based thixotropic gel
and a buffered sodium citrate solution at 0.10 M; and

[0034] a polyethylene terephthalate separator tube containing a highly
thixotropic gel formed by a polymer mixture and an anhydrous sodium
citrate at 3.5 mg/mL;

characterised in that the device has an inlet for introducing said whole
blood, is held in a vacuum intended to aspirate the whole blood sample,
is sterile, has a usable vacuum of or about 8 to about 10 mL and is
suitable for undergoing centrifugation.

[0035] In a sixth aspect, the present invention provides a use of a
platelet concentrate according to the invention for the manufacture of a
medicament for healing of wounds or for promoting bone or periodontum
growth and/or bone and/or tissue regeneration.

[0036] In a seventh aspect, the present invention provides a use of a
platelet concentrate according to the invention for the manufacture of a
cosmetic preparation for use as anti-aging agent or skin repairing agent
such as a scar repairing agent, a wrinkle filling and/or repairing agent.

[0037] In an eighth aspect, the present invention provides a
pharmaceutical composition comprising platelet concentrate according to
the invention and a pharmaceutically acceptable carrier.

[0038] In a ninth aspect, the present invention provides a cosmetic
composition comprising platelet concentrate according to the invention
and a cosmetically acceptable carrier.

[0039] In a tenth aspect, the present invention provides an implantable
device for use in tissue regeneration therapy comprising:

(a) a permeable core comprising a platelet concentrate of the invention;
and (b) an external jacket surrounding said core, said jacket comprising
a biocompatible material, preferably bioresorbable.

[0040] In an eleventh aspect, the invention provides a kit adapted for
tissue regeneration comprising a separator tube according to the
invention, phlebotomy accessories for the preparation of the wound
healant according to the invention and an applicator device (e.g. a
double syringe) for the simultaneous dispensation onto the wound of the
platelet concentrate according to the invention and a coagulation
activator.

[0041] In a twelfth aspect, the invention provides a method for promoting
wound healing and/or sealing and/or tissue and/or bone regeneration in a
wound of a human or a lower animal comprising:

[0042] a) Providing a
wound healant according to the invention;

[0043] b) Applying a
therapeutically effective amount of the said wound healant to a wound, a
damaged tissue or a damaged bone.

[0044] In a thirteenth aspect, the invention provides a method for
inducing periodontal regeneration in a wound or a periodontal defect of a
mammal with periodontal disease or other condition requiring periodontal
regeneration comprising:

a) Providing a wound healant according to the invention; b) Applying a
therapeutically effective amount of the said wound healant to the said
wound or said periodontal defect or cavity; c) Optionally inserting a
periodontal barrier, wherein the barrier is positioned between the
gingival tissue and the wound treated according to steps a) and b) and
the said barrier is selected from a membrane, a biodegradable polymer
and/or a biocompatible porous material; d) Closing the wound.

[0045] In a fourteenth aspect, the invention provides a method for
promoting skin regeneration in a scar or a wrinkle from human or lower
animal comprising:

a) Providing a wound healant according to the invention; b) Filling the
skin scar or wrinkle line with the said wound healant.

[0046] In a fifteenth aspect, the present invention provides a process for
the preparation of a cell composition, comprising the steps of:

[0053] (e) Admixing the platelet concentrate obtained under
step (c) with the cell extract obtained in (d); wherein the
centrifugation step a) is performed at a force of or about 1500 g up to
about 2000 g in a sufficient length of time to form a barrier between the
plasma containing the platelets, the lymphocytes and the monocytes and
the pellet containing the erythrocytes; the separation step b) is made by
collecting the supernatant from atop of said barrier and wherein the
enriched plasma is enriched in leucocytes, thrombocytes and adhesion
proteins (for example, fibronectin) as compared to native whole blood.

[0054] In a sixteenth aspect, the present invention provides a process for
the preparation of a wound or tissue healing composition, comprising the
steps of:

a) Centrifuging whole blood in a separator tube selected from:

[0055] a
glass separator tube containing a polyester-based thixotropic gel and a
buffered sodium citrate solution at 0.10 M; and

[0056] a polyethylene
terephthalate separator tube containing a highly thixotropic gel formed
by a polymer mixture and an anhydrous sodium citrate at 3.5 mg/mL; b)
Optionally separating the enriched platelet rich plasma from the full
plasma by removing half of the supernatant containing the platelet poor
plasma; c) Re-suspending the enriched plasma; d) Admixing the platelet
concentrate obtained under step (c) with a coagulation activator in a
vol. ratio (platelet concentrate:coagulation activator) of about 10:1 up
to about 10:3; e) Providing a cell extract such as an extract of dermal
cells such as keratinocytes, fibroblasts, melanocytes and Langerhans
cell; fat cells; bone marrow cells; muscle cells such as myoblasts and
satellite cells; osteoblasts; chondrocytes; periosteal membrane cells;
corneal cells; umbilical cord cells; Schwann cells, tendon cells or
pancreas islet cells; f) Admixing the platelet concentrate admixture
obtained under step (d) with the cell extract obtained in (e); wherein
the centrifugation step is performed at a force of or about 1500 g up to
about 2000 g in a sufficient length of time to form a barrier between the
plasma containing the platelets, the lymphocytes and the monocytes and
the pellet containing the erythrocytes; the separation step b) is made by
collecting the supernatant from atop of said barrier and wherein the
enriched plasma is enriched in leucocytes, thrombocytes and adhesion
proteins (for example, fibronectin) as compared to native whole blood.

a) plasma; b) platelets at a concentration of at least 300×109
cells/L; c) white blood cells at a concentration of at least
7.0×109 cells/L; d) fibrinogen at a concentration of at least
3 mg/L; e) a cell extract, such as an extract of dermal cells such as
keratinocytes, fibroblasts, melanocytes and Langerhans cell; fat cells;
bone marrow cells; muscle cells such as myoblasts and satellite cells;
osteoblasts; chondrocytes; periosteal membrane cells; corneal cells;
umbilical cord cells; Schwann cells, tendon cells or pancreas islet cells
wherein cells are at a concentration of about 105 to about 106
cells/L or a concentration of about 105 to about 106 cells/ml
of plasma or enriched plasma; and wherein the erythrocyte concentration
is less than 0.6×1012 cells/L.

a) plasma; b) platelets at a concentration of at least 300×109
cells/L; c) white blood cells at a concentration of at least
7.0×109 cells/L; d) fibrinogen at a concentration of at least
3 mg/L; e) coagulation activator in a vol. ratio (platelet
concentrate:coagulation activator) of about 10:1 to about 10:3; f) a cell
extract, such as an extract of dermal cells such as keratinocytes,
fibroblasts, melanocytes and Langerhans cell; fat cells; bone marrow
cells; muscle cells such as myoblasts and satellite cells; osteoblasts;
chondrocytes; periosteal membrane cells; corneal cells; umbilical cord
cells; Schwann cells, tendon cells or pancreas islet cells wherein cells
are at a concentration of about 105 to about 106 cells/L or at
a concentration of about 105 to about 106 cells/ml of plasma or
enriched plasma; and wherein the erythrocyte concentration is less than
0.6×1012 cells/L.

[0059] In a nineteenth aspect, the invention provides a wound or tissue
healing composition comprising an isolated cell composition according to
the invention.

[0060] In a twentieth aspect, the invention provides a method for
promoting wound healing and/or sealing and/or regeneration of a tissue
and/or a cartilage and/or a bone and/or a nerve in a human or a lower
animal comprising:

[0061] a) Providing a wound or tissue healing
composition or a cell composition according to the invention;

[0062] b)
Applying a therapeutically effective amount of the said a wound or tissue
healing composition or cell composition to a wound, a damaged tissue or a
damaged cartilage or a damaged bone.

[0063] In a twenty-first aspect, the invention provides a method for
increasing adipose tissue volume in a mammal with a dermal fat graft or
other condition requiring adipose tissue regeneration comprising:

a) Providing a fat cell composition according to the invention; b)
Applying a therapeutically or cosmetically effective amount of the said
fat cell composition to the dermal fat graft or the adipose tissue
requiring adipose tissue regeneration; c) Optionally inserting a surgical
flap or implant, wherein the surgical flap or implant, is positioned in
the site requiring regeneration or volumetric amplification and the said
surgical flap or implant comprises a combination of a fat cell
preparation according to the invention and plasma or enriched plasma
material.

[0064] In a twenty-second aspect, the invention provides a method for
inducing myocardial regeneration in a mammal with myocardial deficiency
or other condition requiring myocardial regeneration tissue regeneration
comprising:

a) Providing a muscle cell or a bone marrow cell composition according to
the invention; b) Applying a therapeutically effective amount of the said
muscle cell composition to the myocardial tissue requiring regeneration.

[0065] In a twenty-third aspect, the invention provides a method for
inducing corneal regeneration in a mammal with corneal deficiency or
other condition requiring corneal regeneration comprising:

a) Providing a cornea cell composition according to the invention; b)
Applying a therapeutically effective amount of the said corneal cell
composition to the corneal tissue requiring regeneration.

[0066] In a twenty-fourth aspect, the invention provides a method for
inducing articular or cartilage regeneration in a mammal with articular
or cartilage deficiency or other condition requiring articular or
cartilage tissue regeneration comprising:

a) Providing a chondrocyte cell or bone marrow cell composition according
to the invention; b) Applying a therapeutically effective amount of the
said chondrocyte cell composition to the articular or cartilage tissue
requiring regeneration; c) Optionally inserting a surgical flap or
implant, wherein the surgical flap or implant, is positioned in the
defect of the cartilage or under a periosteal patch, and the said
surgical flap or implant comprises a combination of a chondrocyte or bone
marrow cell composition according to the invention and plasma or enriched
plasma material.

[0067] In a twenty-fifth aspect, the invention provides a method for
promoting skin regeneration in a scar, a wrinkle or a fat deficiency from
human or lower animal comprising:

a) Providing a wound or tissue healant or a cell composition according to
the invention; b) Filling the skin scar, wrinkle line or fat deficiency
with the said wound or tissue healant or cell composition according to
the invention.

[0068] In a twenty-sixth aspect, the invention provides a method for
inducing peripheral nerve regeneration in a mammal with peripheral nerve
damage, nerve suture or spinal cord injury or other condition requiring
peripheral nerve regeneration comprising:

a) Providing a Schwann cell composition according to the invention; b)
Applying a therapeutically effective amount of the said Schwann cell
composition to the peripheral nerve requiring regeneration.

[0069] In a twenty-seventh aspect, the invention provides a method for
inducing bone regeneration in a mammal with bone damage, bone deficiency
or other condition requiring bone regeneration comprising:

a) Providing a bone marrow cell or osteoblast cell composition according
to the invention; b) Applying a therapeutically effective amount of the
said bone marrow cell or osteoblast cell composition to the bone
requiring regeneration.

[0070] In a twenty-eighth aspect, the invention provides a method for the
treatment of type I diabetes, insulin-dependent diabetes or
hyperglycaemia in a mammal comprising:

a) Providing a pancreas islet cell composition according to the
invention; b) Applying a therapeutically effective amount of the said
pancreas islet cell composition to the patient, for example by injection.

[0071] In a twenty-ninth aspect, the invention provides a method for the
treatment of urinary incontinence in a mammal or other condition
requiring bladder regeneration comprising:

a) Providing a myoblast cell composition according to the invention; b)
Applying a therapeutically effective amount of the said myoblast cell
composition to the bladder neck requiring regeneration.

[0072] In a thirtieth aspect, the invention provides a method for the
treatment of anal incontinence in a mammal or other condition requiring
anal muscle regeneration comprising:

a) Providing a myoblast cell composition according to the invention; b)
Applying a therapeutically effective amount of the said myoblast cell
composition to the para-anal area requiring regeneration.

[0073] In a thirty-first aspect, the invention provides a method for the
treatment of reflux oesophagitis or gastro-oesophageal reflux disorders
in a mammal or other condition requiring oesophageal sphincter
regeneration comprising:

a) Providing a myoblast cell composition according to the invention; b)
Applying a therapeutically effective amount of the said myoblast cell
composition to the oesophageal sphincter requiring regeneration.

[0074] In a twenty-second aspect, the present invention provides a use of
a cell preparation according to the invention for the manufacture of a
medicament for healing of wounds or tissues or for promoting bone or
periodontum growth and/or bone and/or tissue regeneration such as skin,
cartilage, muscle, tendon, adipose tissue, cornea, peripheral nerves,
spine or bone regeneration.

[0075] In a twenty-third aspect, the present invention provides a use of a
cell composition according to the invention for the manufacture of a
cosmetic preparation for use as anti-aging agent or skin repairing agent
such as scar repairing agent, lipoatrophy repairing agent or wrinkle
filling and/or repairing agent.

[0076] In a twenty-fourth aspect, the present invention provides a
pharmaceutical composition comprising a cell composition according to the
invention and a pharmaceutically acceptable carrier.

[0077] In a twenty-fifth aspect, the present invention provides a cosmetic
composition comprising cell composition according to the invention and a
cosmetically acceptable carrier.

[0078] In a twenty-sixth aspect, the present invention provides an
implantable device for use in tissue regeneration therapy comprising:

a) a permeable core comprising a cell composition of the invention; and
b) an external jacket surrounding said core, said jacket comprising a
biocompatible material, preferably bioresorbable.

[0079] The uses, methods and compositions according to the invention are
useful in the regeneration and/or rejuvenation of tissues, bones and/or
cartilages. The uses, methods and compositions according to the invention
are particularly useful in the treatment of diabetic neuropathic ulcers
or decubitus sores; bone and cartilage damages such as deep joint
cartilage or chondral damages such as surgical repair of torn tendons;
arthritis in joint caused by traumas or by aging; rotator cuff disorders;
non-healing wounds such as non-healing wounds such as vasculitis induced
wounds, for example in lower equine limb; periodontal diseases; implant
surgery; cardiac muscle damages such as in chronic cardiac failure, heart
failure, ischemic and non-ischemic disorders, cardiomyopathy;
gastro-oesophageal reflux disease; anal or urinary incontinence; facial
surgery such as facial surgery induced alopecia (alopecia due to hair
follicle loss in the side burn areas), face-lift surgery (rhytidectomy),
rhinoplasty, dermal fat grafts (in the treatment of facial augmentation,
congenital hemiatrophy of the face such as congenital cartilage nose
atrophy and lipoatrophy such as in HIV/AIDS suffering patients, erosion
and arthroscopy); wound healing complications such as after eyelid
blepharoplasty; corneal disorders such as corneal opacity such as those
caused by chemical burns, affliction by Steven's Johnson syndrome and
corneal ulcers; scarring of the cornea; dry eye syndrome; haematological
diseases such as Thalassaemia; peripheral nerve damage, nerve suture and
spinal cord injury; bone defects or disorders such as bone graft or bone
fracture, skin damages or disorders such as acne (especially after
dermabrasion treatment), burns, rubella or small pox scars, vitiligo,
lipoatrophy, Kaposi's sarcoma, skin skeloids or Dupuytren's palmar
fibromatosis.

[0080] In another aspect, the uses, methods and compositions according to
the invention are useful in the regeneration and/or rejuvenation of skin
tissues, particularly in promoting and/or initiating skin regeneration
such as reducing skin wrinkles, acne (especially after dermabrasion
treatment), burns, rubella or small pox scars, vitiligo and lipoatrophy
(e.g. anti-aging compositions and skin regeneration compositions),
amelioration of nasolabial lines and treatment of skin damages or
disorders such as skin burns, Kaposi's sarcoma, skin skeloids or
Dupuytren's palmar fibromatosis and in the reduction of pain associated
with skin and tissue regeneration.

DESCRIPTION OF THE FIGURES

[0081] FIG. 1 is a schematic representation of the variation of
concentration in growth factors (PDGF-AB, EGF and VEGF) of a platelet
concentrate composition according to the invention versus time (T in
hours) after the centrifugation step in the preparation process of the
invention.

[0082] FIG. 2 is a schematic representation of the outcome of the
treatment of a skin graft donor site with a preparation containing a
platelet concentrate composition according to the invention in comparison
with a control group in terms of healing time in days (HT), pain at day 5
on a scale 0 to 10 (P) and epithalization at day 5 on a scale 0 to 7 (E).
Control group: C, platelet-rich preparation alone: RegenPRP®,
platelet-rich preparation and autologous keratinocytes:
RegenExtracell®. The dotted line indicates when the first bandage is
changed at day 5.

[0083] FIG. 3 represents the morphology of a human fibroblast expanded in
cell preparation according to the invention under the condition described
in Example 7, showing branching and filopodia ×5,000 (Olympus®
inverted microscope).

[0084]FIG. 4a represents a 3D scaffold of human fibroblasts from a cell
preparation according to the invention under the condition described in
Example 7.

[0085]FIG. 4b represents monolayers of fibroblasts densely packed in 3D
culture from a cell preparation according to the invention under the
condition described in Example 7.

DETAILED DESCRIPTION OF THE INVENTION

[0086] The following paragraphs provide definitions of the terms according
to the invention and are intended to apply uniformly throughout the
specification and claims unless an otherwise expressly set out definition
provides a broader definition.

[0087] By the expression "thixotropic" is meant a gel that becomes more
fluid as a result of agitation or pressure, i.e. a gel which viscosity is
decreasing as a result of agitation or pressure. The term viscosity
refers to those characteristics of the specified material(s) determining
the degree of gelation, such as for example the firmness or hardness of
the material, the degree to which the material resists flowing like a
fluid. A thixotropic gel according to the invention comprising a
polyester gel or a mixture thereof which is water insoluble and
chemically inert to blood constituents which can be used in accordance
with the invention. Typical thixotropic gels are used in blood cells
separation for diagnostics and proteomics purposes.

[0088] By the expression "point-of-care" is meant all services provided to
patients at the bedside.

[0089] By the expression "phlebotomy accessories" or "venipuncture
accessories" is meant accessories that allow the puncture of a vein with
a needle for the purpose of drawing blood.

[0090] By the expression "wound healant" or "wound sealant" or a "tissue
healing composition" is meant an agent or a composition that is able to
promote and/or increase the speed and/or quality of cicatrisation of a
wound. Wound healants or sealants are able to promote tissue
regeneration.

[0091] By the expression "wound" is meant any damaged tissue, for example
following trauma or surgery. Wounds in mammals, include for examples bed
sores, ulcers, lacerations and burns, graft sites (graft donor and
acceptor sites), fistulas, periodontal tissue damages, diabetic
non-healing wounds, consequences of traumas or any surgery act. In its
general sense the expression is intended to also encompass skin damages
where the skin surface presents some depression without necessarily a cut
on its surface such as age-related tissue damages (e.g. wrinkles) and
scars such as for example acne (especially after dermabrasion treatment)
or rubella scars.

[0092] By the expression "PRP" is intended to mean a platelet-rich-plasma,
preferably of human origin, more preferably autologous, prepared by the
process of the invention in order to pellet and remove erythrocytes and
concentrate the plasma in leucocytes, thrombocytes and adhesion proteins
as compared to native whole blood.

[0093] By the expression "autologous" or "autogenic" or "autogenous" is
intended a method of the invention using a single donor's blood and
wherein the blood extracted from this donor is intended for use on the
same donor. As opposed, "allogeneic" methods are using blood from one or
more third parties for use on a donor ("homologuous" or "heterologuous").
An autologous product avoids some of the common problems associated with
the use of biological materials from third parties, such as for example
screening to assure that the donor was biologically or immunologically
compatible with the patient and potential contamination with hepatitis,
HIV, prion, Creutzfeld-Jacob's disease and the like.

[0094] By the expression "coagulation activator" is intended an agent that
is able to trigger or activate coagulation. A coagulation activator
comprises a thrombin activator and/or a fibrinogen activator.

[0095] By the expression "thrombin activator" is intended an agent that is
able to activate thrombin and to trigger coagulation. Typical thrombin
activators are certain cofactors such as sodium or calcium. In practicing
this invention, thrombin activation preferably occurs in the presence of
calcium ions. Calcium ions are generally added to the platelet
concentrate as a salt solution to provide a final concentration generally
of or about 0.1 mg/mL of platelet concentrate. Suitable calcium salts
include, without limitation, CaCO3, and CaSO4. A preferred
calcium salt for use in the invention is calcium chloride (CaCl2).
CaCl2 is available as calcium chloride injection, USP 10% (Regen
Lab, Switzerland).

[0096] By the expression "fibrinogen activator" is intended an agent that
is able to activate the conversion of fibrinogen into fibrin and triggers
the formation of the clot. Typical fibrinogen activators are thrombin or
batroxobin. The term thrombin may include calcified thrombin, in
particular, from or about 100 to about 10 units of thrombin per 1 mL of
10% of aqueous calcium chloride solution; it may include calcified bovine
thrombin, allogeneic thrombin or recombinant human thrombin, preferably
autologous thrombin. A fibrinogen activator can be an enriched thrombin
composition such as thrombin compositions as described in U.S. Pat. No.
6,472,162 or an autologous thrombin serum according to the invention.

[0097] By the expression "therapeutically effective amount" is intended
the amount or amounts of the constituent elements or combination thereof
necessary to enhance wound healing such as, for example, the reduction in
the volume or surface area of a wound, the increase in the amount of
granulation tissue or other biological material facilitating collagen lay
down, vascular in growth, fibroblast proliferation or overall healing;
All of the versions of the invention described herein are assumed to have
the therapeutically effect amount(s) of constituent substances, or
combinations thereof.

[0100] The compositions, uses and methods according to the invention are
particularly useful in wound or tissue healing or regeneration
treatments, especially the treatment of traumatic or surgical wounds such
in the fitting and/or holding and/or sealing of native or prosthetic
grafts (especially skin, bone grafts and/or dental prostheses or implants
or the like, including also the graft donor site); treatment of
vasculitis; ulcers such as diabetic neuropathic ulcers or decubitus
sores; radiodermatitis (e.g. after irradiation on an epidermoidal skin
carcinoma) and closing fistulas (such as for cyclists).

[0101] Further, the compositions, uses and methods according to the
invention are particularly useful in the treatment of cardiac disorders,
cardiac regeneration such as in the treatment of heart failure, chronic
cardiac failure, ischemic and non-ischemic cardiac failure and
cardiomyopathy.

[0102] Further, the compositions, uses and methods according to the
invention are particularly useful in the treatment of urinary and/or anal
incontinence.

[0103] Further, the compositions, uses and methods according to the
invention are particularly useful in the treatment of reflux oesophagitis
and/or gastro-oesophageal reflux disorder.

[0104] Further, the compositions, uses and methods according to the
invention are particularly useful in the treatment of skin damages such
as in skins damaged by radiations (radiodermatitis or sun damaged skin),
aged skins or burned skins and/or in the amelioration of facial wrinkles,
rhytids, acne (especially after dermabrasion treatment), burns, rubella
or small pox scars, vitiligo, lipoatrophy or lipodystrophy, Kaposi's
sarcoma, skin skeloids or Dupuytren's palmar fibromatosis and/or in skin
rejuvenation treatments.

[0105] Further, the compositions, uses and methods according to the
invention are particularly useful in the treatment of lipoatrophy such as
in HIV/AIDS patients and in other congenital hemiatrophy of the face such
as congenital cartilage nose atrophy.

[0106] Further, the compositions, uses and methods according to the
invention are particularly useful in the treatment of bone, cartilage and
articular disorders such as chondral damage, arthritis, cartilage and/or
bone injury such as deep cartilage damage and/or erosion and/or
arthroscopy, tendon torn and rotator cuff in shoulder.

[0107] Further, the compositions, uses and methods according to the
invention are particularly useful in the treatment of hematological
diseases such as Thalassaemia.

[0108] Further, the compositions, uses and methods according to the
invention are particularly useful in the treatment of corneal disorders
such as dry eye syndrome; corneal opacity such as those caused by
chemical burns, affliction by Steven's Johnson syndrome; scarring of the
cornea and corneal ulcers.

[0109] Further, the compositions, uses and methods according to the
invention are particularly useful in the treatment of peripheral nerve
damage, nerve suture and spinal cord injury.

[0110] Further, the compositions, uses and methods according to the
invention are particularly useful in the treatment of type1 diabetes,
insulin-dependent diabetes and/or hyperglycaemia.

[0111] Further, the compositions, uses and methods according to the
invention are particularly useful in the treatment of bone defects or
disorders such as bone graft or bone fracture.

[0112] The use of the resulting composition the invention can be further
modified before application and according to the therapeutic objective.

[0113] Compositions of the invention can be used together with bone
filling materials, especially resorbable filling materials such as
hydroxyapatite (calcium phosphate ceramic used as a biomaterial) or
demineralised bone, or used as a mixture with bone extracts in a process
for the regrowth of bone for example in craniofacial and orthopaedic
procedures.

[0114] Compositions of the inventions may be used as a wound sealant in
plastic surgery including burn grafting and other free skin graft
applications, for example in oncology for favouring tissue regeneration,
including speeding (neo)vascularization.

[0115] The compositions according to the invention are particularly useful
in wound healing treatments at the skin graft donor site. The removal of
a skin graft on a healthy skin creates a new wound at the donor's site
which normally heals spontaneously between 12 to 14 days. However, this
cicatrisation is extremely demanding for the body, especially if the
donor site is broad or the person is less resistant (e.g. burn victims,
people suffering from multiple traumas, people treated with corticoids,
children or elderly) and the energetic losses are even increased by the
loss in minerals, trace elements and proteins induced by the fluid losses
from the new wound. In addition, important pain during the first 8 days
is often present on the graft donor's site. Pain reduction treatments are
often used such as the use of analgesics (e.g. morphine) and/or
hydrocellular wound dressings, however pain remains present, especially
during the dressing change that occurs imperatively within 48 hours up to
1 week after the graft removal. In addition, the hydrocellular wound
dressings have the drawbacks not only to be rather expensive but also by
maintaining humidity on the wound, to prevent its drying, to increase the
wound deepness, to favour the outbreak of bacterial infections and to
lead to non-esthetic scars. Therefore, a stimulation of the skin graft
donor site healing is very desirable.

[0116] Compositions of the invention are particularly adapted to chronic
wounds that may lack sufficient blood circulation to facilitate the wound
healing cascade.

[0117] The compositions and methods according to the invention may be also
used in the treatment of periodontal disease where a loss and/or a damage
of the periodontal tissues is observed, such a treatment comprising for
example placing at the periodontal site or cavity in a human or a lower
animal in need of periodontal tissue regeneration a composition according
to the invention.

[0118] The compositions according to this invention are effective in
eliminating or greatly reducing post-operative bleeding and extravasation
or loss of serous or other fluid in these applications, in reducing the
infection risk caused by most bacteria and/or enhances connective tissue
formation compared to natural healing (i.e. no exogenous agents added) or
to healing obtained through the use of other platelet concentrates
prepared with known methods.

[0119] The compositions according to the invention are particularly useful
in the preparation of pharmaceutical for promoting and/or initiating
wound healing and/or tissue regeneration or for the preparation of
cosmetic compositions for skin regeneration such as reducing skin
wrinkles, acne (especially after dermabrasion treatment), rubella or
small pox scars, vitiligo and lipoatrophy (e.g. anti-aging compositions
and skin regeneration compositions).

[0120] The compositions of the present invention may be administered
locally or injected in the wound or in or near to the grafted organ or
injected subcutaneously. Local administration may be by injection at the
site of injury or defect or by insertion or attachment of a solid carrier
at the site, or by admixture with a cream or emulsion, or by inclusion in
a tissue or paper or hydrogel carrier, or by direct, topical application
of the composition of the invention such as in the form of eye drops.
Preferably, the compositions are readily syringable compositions. The
mode of administration, the dosage administered, as single or multiple
doses, to an individual will vary depending upon a variety of factors,
including pharmacokinetic properties, patient conditions and
characteristics (sex, age, body weight, health, size), extent of
symptoms, concurrent treatments, frequency of treatment and the effect
desired.

[0121] The compositions of the present invention may be administered in
combination with a co-agent useful in the treatment of tissue
regeneration such as a healing agent, a wrinkle filler, an anti-aging
agent such as an anti-aging vitamin complex, an antibacterial agent,
antibiotic agent, an corticosteroid agent, an antalgic and analgesic
agent, or an anesthetic agent like adrenaline, etc. . . . The invention
comprises compositions combined with a co-agent useful in the treatment
of tissue regeneration for simultaneous, separate or sequential use in
tissue regeneration therapy such as wound healing, bone and periodontum
growth repair.

[0122] The compositions of the invention, the device and procedures for
the preparation of autologous platelet concentrates or cell compositions
of the invention are particularly useful for therapeutic use,
particularly as autogenous biological glue in a haemostatic system
intended to accelerate the physiological process of tissue regeneration,
for example in dental implantology, skin and bone surgery, cartilage and
tendon surgery, corneal and peripheral nerve regeneration and cardiac
surgery. The compositions of the invention, the device and procedures for
the preparation of autologous platelet concentrates and cell composition
of the is invention are particularly useful for cosmetic use,
particularly as autogenous rejuvenation material intended to be used for
example as wrinkle, scar or fat deficiency filler, alone on in
combination with at least one anti-aging agent.

[0123] The platelet concentrate of the invention may be combined with an
autologous cell extract preparation such as for example keratinocytes,
bone marrow cells, osteoblasts, chondrocytes, fibroblasts, periosteum,
melanocytes and Langerhan's cell; fat cells; bone marrow cells; muscle
cells such as myoblasts and satellite cells; periosteal membrane cells;
corneal cells; umbilical cord cells; tendon cells or pancreatic islet
cells. Keratinocytes can be harvested through a method described by
Reinwald and Green, 1975, Cell, 6(3):331-43. Other mentioned cells can be
harvested through methods described in "Culture de cellules animales;
methologies-applications", 2003, Ed. Barlovatz-Meimom and Adolphe, INSERM
editions, Paris. Alternatively, cell extracts are derived from a cell
bank or a cell culture or harvested as described in the Examples below.

[0124] The platelet concentrate and cell compositions of the invention
have proven to be really beneficial in the acceleration and/or promotion
of the healing process of wounds, even chronic unhealing wounds, leading
to successful closures where weeks of conventional therapies had failed
and achieving a decrease in infection risks, an improvement in patient's
recover and comfort, a reduction of medical care costs and a better
esthetic final result.

[0125] The compositions of the invention can of course be also prepared
from plasma derived from several identified donors. The invention is not
limited to autologous biological materials, such as collection of
concentrated platelets from the wounded's own biological material. The
invention encompasses the use of biological materials obtained from one
or more third parties, who need not be of the same species as the patient
whose wound is being treated with the wound healant composition described
herein unless bio-incompatibility would result from the use of such third
party biological materials.

[0126] In one embodiment, the invention provides a process for the
preparation of a platelet concentrate composition or a cell composition
as described herein.

[0127] In another embodiment, the present invention provides a device for
the preparation of a platelet concentrate composition from whole blood as
described herein.

[0128] In a further embodiment, is provided by the invention a process for
the preparation of a platelet concentrate composition wherein the
centrifugation step is performed at force between about 1,500 g and up to
about 1,700 g for a time selected from about 3 min up to about 15 min,
preferentially at 1,500 g for about 8 min.

[0129] In another further embodiment, is provided by the invention a
process for the preparation of a platelet concentrate composition wherein
the separator tube has an inlet for introducing said whole blood, is held
in a vacuum intended to aspirate the whole blood sample, is sterile, has
a usable vacuum of 8 to 10 mL and is suitable for undergoing
centrifugation.

[0130] In another further embodiment, is provided by the invention a
process for the preparation of a platelet concentrate composition wherein
the separator tube is a polyethylene terephthalate separator tube
containing a highly thixotropic gel formed by a polymer mixture and an
anhydrous sodium citrate at 3.5 mg/mL.

[0131] In another embodiment, the present invention provides an isolated
platelet concentrate composition obtainable from the process according to
the invention.

[0132] In another embodiment, is provided by the invention an isolated
platelet concentrate composition comprising:

a) plasma; b) platelets at a concentration of at least 300×109
cells/L, preferably of at least 350×109 cells/L, more
preferably of at least 400×109 cells/L; c) white blood cells
at a concentration of at least 7.0×109 cells/L, preferably of
at least 8.0×109 cells/L; d) fibrinogen at a concentration of
at least 3 mg/L; and wherein the erythrocyte concentration is less than
0.6×1012 cells/L, preferably less than 0.5×1012
cells/L.

[0133] In another embodiment, the present invention provides a wound or
tissue healant composition comprising:

a) plasma; b) platelets at a concentration of at least 300×109
cells/L, preferably of at least 350×109 cells/L, more
preferably of at least 400×109 cells/L; c) white blood cells
at a concentration of at least 7.0×109 cells/L, preferably of
at least 8.0×109 cells/L; d) fibrinogen at a concentration of
at least 3 mg/L; e) a coagulation activator in a vol. ratio (platelet
concentrate:coagulation activator) of about 10:1 to about 10:3; f)
optionally an autologous cell extract, such as extract of keratinocytes,
bone marrow cells, fibroblasts, periosteum, melanocytes and Langerhans
cell; fat cells; bone marrow cells; muscle cells such as myoblasts and
satellite cells; osteoblasts; chondrocytes; periosteal membrane cells;
corneal cells; umbilical cord cells; tendon cells or pancreatic islet
cells; and wherein the erythrocyte concentration is less than
0.6×1012 cells/L, preferably less than 0.5×1012
cells/L.

[0134] In another embodiment, is provided by the invention a process for
the preparation of a wound or tissue healant composition as described
herein.

[0135] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant composition
wherein the coagulation activator which is admixed is 10% calcium
chloride.

[0136] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant composition
wherein the coagulation activator which is admixed under step b) is a
thrombin enriched preparation. A method for preparing thrombin for use in
a biological glue is described in U.S. Pat. No. 6,472,162 by the addition
of 8 to 20% ETOH to a volume of plasma and this preparation may be used
as a thrombin enriched preparation in the context of the invention.
Alternatively, an autologous thrombin serum (ATS) can be used a thrombin
enriched preparation in the context of the invention. An autologous
thrombin serum according to the invention is obtained by a process
comprising (i) the addition to a patient's whole blood sample (e.g. 8 mL)
collected in a separator tube of the invention, a 10% of final volume of
calcium chloride 10% (e.g. 1 mL) and a 10% of the final volume of a
preparation of 95% v. ethanol solution (e.g. 1 mL) and (ii) precipitation
for about 30 min at room temperature. After 30 min, a centrifugation at
or about 1,500 g for about 8 to 10 min. In a further preferred
embodiment, the thrombin enriched preparation and preferably the
autologous thrombin serum is admixed under step b) directly on the wound.

[0137] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant composition
according to the invention wherein a further step b') wherein the
activated platelet-rich preparation composition (obtained by the admixing
of the platelet concentrate with the said coagulation activator) obtained
in step b) may be partially dehydrated by the contact of a wound dressing
covered by a soft hydrophobic layer to avoid contamination with
micro-strings from the dressing in order to obtain a semi-solid gel that
can be manipulated by appropriate instruments, for example to fill a
cavity or tissue deficiency, or as a growth matrix ("scaffold") while
waiting for the reconstitution of the autogenous extracellular matrix.
The obtained wound or tissue healant is particularly useful in a method
for inducing periodontal regeneration in a wound, a tissue or a
periodontal defect or a cavity.

[0138] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant or cell
composition wherein the cell extract is an extract of keratinocytes.

[0139] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant or cell
composition wherein the cell extract is an autologous extract of
keratinocytes.

[0140] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant or cell
composition wherein the cell extract is an extract of skeletal muscle
cells such as muscle progenitor cells or satellite stem cells.

[0141] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant or cell
composition wherein the cell extract is an extract of fibroblasts.

[0142] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant or cell
composition wherein the cell extract is an extract of adipocytes.

[0143] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant or cell
composition wherein the cell extract is an extract of chondrocytes.

[0144] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant or cell
composition wherein the cell extract is an extract of stem cells such as
umbilical cord stem cells.

[0145] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant or cell
composition wherein the cell extract is an extract of tendon cells.

[0146] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant or cell
composition wherein the cell extract is an extract of periosteal membrane
or gingival cells.

[0147] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant or cell
composition wherein the cell extract is an extract of corneal cells.

[0148] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant or cell
composition wherein the cell extract is an extract of bone marrow cells.

[0149] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant or cell
composition wherein the cell extract is an extract of osteoblast cells.

[0150] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant or cell
composition wherein the cell extract is an extract of Schwann cells.

[0151] In another further embodiment, is provided by the invention a
process for the preparation of a wound or tissue healant or cell
composition wherein the cell extract is an extract of pancreas islet
cells.

[0152] In another further embodiment, the isolated platelet concentrate
composition, the wound or tissue healant composition, the thrombin
enriched serum and/or the cell extract of the invention is/are
autologous.

[0153] In a further aspect, the present invention provides a kit adapted
for tissue regeneration according to the invention wherein the kit
further comprises separate vials containing ETOH and CaCl2, syringe
holders, clumper and a tip applicator with a dual exit.

[0154] In a further aspect, present invention provides a kit adapted for
tissue regeneration according to the invention comprising two sterile
blisters:

[0155] (1) one blister comprising accessories for the
phlebothomy, separator tubes of the invention, vials of ETOH and
CaCl2 for the preparation of an autologous thrombin serum; and

[0156] (2) a second blister comprising accessories for two syringe
holders and clumper, and tip applicator with a dual exit.

[0157] In another embodiment, the invention provides a process for the
preparation of a cell composition according to the invention wherein the
cell extract provided under step d) or e) is obtained by a process
comprising the steps of:

(A) Providing the said cells in a platelet concentrate according to the
invention; (B) Optionally culturing the cells; (C) Re-suspending the
cultured cells obtained under step (B) into a platelet concentrate
according to the invention.

[0158] In a further embodiment, the invention provides a process for the
preparation of a cell composition according to the invention wherein the
cell expansion under step (A) is performed in a platelet concentrate
according to the invention such as the final concentration in platelets
comprised between about 5% and about 40% of the volume of the culture
medium.

[0159] In another further embodiment, the invention provides a process for
the preparation of a cell composition according to the invention wherein
the cell culture step (B) comprises at least one step of plating the
cells, for example on a cell culture surface such as a Petri dish or a
culture flask.

[0160] In another further embodiment, the invention provides a process for
the preparation of a cell composition according to the invention
comprising at least one further step of harvesting the cells after the
cell culture step (B).

[0161] In another further embodiment, the invention provides a process for
the preparation of a cell composition according to the invention wherein
the cell culture step (B) is performed at 37° C.

[0162] In another further embodiment, the invention provides a process for
the preparation of a cell composition according to the invention wherein
the cell culture step (B) is performed under a gas flow comprising oxygen
or air and carbon dioxide, typically the gas flow comprises 95% of oxygen
or air and 5% carbon dioxide.

[0163] In another further embodiment, the invention provides a process for
the preparation of a cell composition according to the invention wherein
the cell culture step (B) is lasting for about 3 up to about 4 weeks.

[0164] In another further embodiment, the invention provides a process for
the preparation of a cell composition according to the invention wherein
during the cell culture step (B), the cell culture medium is regularly
changed during incubation, typically every about 3 days.

[0165] In another further embodiment, the invention provides a process for
the preparation of a cell composition according to the invention wherein
the cell culture step (B), comprises at least one exposure step of the
cells to visible light, typically at about 633 nm, during about 10
minutes. In another aspect, the exposure step to visible light is
repeated once a week during cell incubation.

[0166] In another further embodiment, the invention provides a process for
the preparation of a cell composition according to the invention wherein
the cell composition is a keratinocyte or fibroblast cell composition and
the cell culture step (B), comprises at least one exposure step of the
cells to visible light, typically at about 633 nm, during about 10
minutes. In another aspect, the exposure step to visible light is
repeated once a week during cell incubation.

[0167] In another further embodiment, the invention provides a process for
the preparation of a cell composition according to the invention wherein
the cell culture step (B), comprises at least one step of addition of
diluted platelet concentrate according to the invention such as the final
concentration in platelets comprised between about 5% and about 40% of
the volume of the culture medium

[0168] In another further embodiment, the invention provides a process for
the preparation of a cell composition according to the invention wherein
the cell composition is a keratinocyte or fibroblast cell composition and
the cell culture step (B), comprises at least one step of addition of
diluted platelet concentrate according to the invention such as the final
concentration in platelets comprised between bout 5% and about 40% of the
volume of the culture medium.

[0169] In another embodiment, the present invention provides an isolated
cell composition obtainable from a process according to the invention.

[0170] In another embodiment, the present invention provides an isolated
cell composition, wherein the isolated cell composition is a fat cell
composition such as an adipocyte cell composition.

[0171] In another embodiment, the present invention provides an isolated
cell composition, wherein the isolated cell composition is a muscle cell
composition such as a myoblast cell or a satellite stem cell composition.

[0172] In another embodiment, the present invention provides an isolated
cell composition, wherein the isolated cell composition is a corneal cell
composition.

[0173] In another embodiment, the present invention provides an isolated
cell composition, wherein the isolated cell composition is a cartilage
cell composition, such as a chondrocyte cell composition.

[0174] In another embodiment, the present invention provides an isolated
cell composition, wherein the isolated cell composition is a skin cell
composition, such as a fibroblast cell or keratinocyte cell composition.

[0175] In another embodiment, the present invention provides an isolated
cell composition, wherein the isolated cell composition is a periosteal
membrane or gengival cell composition.

[0176] In another embodiment, the present invention provides an isolated
cell composition, wherein the isolated cell composition is a tendon cell
composition, such as tendon cell composition.

[0177] In another embodiment, the present invention provides an isolated
cell composition, wherein the isolated cell composition is a stem cell
composition, such as an umbilical cord stem cell composition.

[0178] In another embodiment, the present invention provides an isolated
cell composition, wherein the isolated cell composition is a bone marrow
cell composition.

[0179] In another embodiment, the present invention provides an isolated
cell composition, wherein the isolated cell composition is a Schwann cell
composition.

[0180] In another embodiment, the present invention provides an isolated
cell composition, wherein the isolated cell composition is a pancreas
islet cell composition.

[0181] In another embodiment, the present invention provides an isolated
cell composition, wherein the isolated cell composition is an osteoblast
cell composition.

[0182] In another embodiment, the present invention provides an isolated
cell composition, wherein cells are at a concentration of about
3×105 to about 106 cells/ml of plasma or enriched plasma.

[0183] In another embodiment, the present invention provides compositions,
methods and uses for promoting wound sealing and/or tissue and/or bone
regeneration in a wound of a human or a lower animal as described herein.

[0184] In another further embodiment, the present invention provides
compositions, methods and uses for promoting wound sealing and/or tissue
and/or bone regeneration in a wound of a mammal, preferably human.

[0185] In another embodiment, the present invention provides compositions,
methods and uses for inducing periodontal regeneration in a wound or a
periodontal defect of a mammal with periodontal disease or other
condition as described herein.

[0186] In another further embodiment, the present invention provides a
method for inducing periodontal regeneration in a wound or a periodontal
defect or cavity of a mammal with periodontal disease or other condition
wherein the mammal is human.

[0187] In another further embodiment, the present invention provides a
method for inducing periodontal regeneration in a wound or a periodontal
defect or cavity according to the invention wherein the said
therapeutically effective amount of the said wound or tissue healant
composition is applied in a form of semi-solid gel or a growth matrix to
the said wound or said periodontal defect or cavity such as described for
example in Garg et al., is 2000, Dental Implantology Update, 11(6),
41-44.

[0188] In another embodiment, the present invention provides a method for
promoting skin tissue regeneration in a scar or wrinkle as described
herein.

[0189] In another embodiment, the present invention provides a method for
inducing myocardial regeneration according to the invention, wherein the
said therapeutically effective amount of the said muscle cell composition
according to the invention is injected into the myocardium, typically
into the left ventricule myocardium. Injection can be made as direct
injection or multiple catheter injection. Myoblasts or satellite cells
can be engineered ex vivo as described in the present description onto a
de-epithelised and UV irradiated human biological amnion and biocomposite
construct, as a monolayer in the present description. The amnion is then
sutured to the ischaemic epicardium in order to repopulate the underlying
tissue with stem cells, in order to improve the contractile power of the
ventricular wall and myocytes.

[0190] In another embodiment, the present invention provides a method for
inducing myocardial regeneration according to the invention, wherein the
said therapeutically effective amount of the said muscle cell composition
according to the invention is injected into the myocardium, together with
a therapeutically effective amount of fibroblast cell composition
according to the invention such as the ratio fibroblast/myoblast is of
about 30:70.

[0191] In another embodiment, the present invention provides a method for
inducing myocardial regeneration according to the invention, wherein the
said therapeutically effective amount of the said muscle cell composition
according to the invention is applied on the ventricular surface in the
form of an amnion patch preferably incubated into a myoblast and
satellite stem cell composition according to the invention.

[0192] In another embodiment, the present invention provides a method for
inducing corneal regeneration according to the invention, wherein the
said therapeutically effective amount of the said corneal cell
composition according to the invention is applied to the corneal tissue
in the form of an amnion patch preferably spread on a dissolvable contact
lens.

[0193] Said method of treating a wound, a tissue or a disease may include
the use of any of the compositions described herein; it may also include
the use of any composition made by any of the methods described herein.

[0194] The methods, the devices and the kit according to the invention
present the advantages to provide a time-effective and relatively
low-cost way of obtaining platelet concentrates in a single operation
that is easy to implement and adapted to a point-of-care application. The
methods of the invention present the advantage to not only lead to
enriched preparations wherein the platelet are concentrated in such a
high yield that is not obtained by known methods but also wherein the
content in erythrocytes is much lower that those obtained by known
methods for the preparation of PRP. The compositions of the invention
present the advantage of having a higher content in platelets, a lower
content in erythrocytes than PRP obtained by known methods and completely
maintained properties for its subsequent therapeutic use in-vivo. More
specifically, the ability of the platelets to release the principal
growth factors involved in tissue regeneration (PDGF, TGF-β, IGF,
VEGF and EGF) at levels for several days (or the 7-10 day life span of
thrombocytes) is maintained.

[0195] In addition, to the extent the compositions of the invention are
made from autologous blood, the invention described herein reduces the
disease transmission and immunoreaction risks associated with the use of
the treatment materials made from biological materials obtained from one
or more third parties.

[0196] The invention therefore provides an improved biological wound
healing and tissue regenerating material, preferably autologous,
promoting tissue such as skin, cartilage and bone regeneration,
especially cicatrisation and/or rejuvenation. The benefits of the
invention comprise a simple and rapid method of preparation of improved
wound healing and tissue regenerating materials adapted to point-of-care
services and which proved to decrease the healing time, associated pain
and medical costs. Further, the wound healing and tissue regenerating
material decreases the graft rejection risks and improves graft success
rates. Further, the improved wound healing and tissue regenerating
materials lead to scars having a much better aesthetic final aspect and
to the durable filling of scars and wrinkles.

[0197] Typically, cell extracts are obtained from a tissue biopsy wherein
the biopsy is preferably performed the day before the mixture with the
platelet concentrate under step a). The size of the biopsies is adapted
to the aimed therapeutic purpose and the types of cells used in the
preparation of the cell composition according to the invention. Examples
of biopsies are given in the Examples below for different types of
tissues.

[0198] Examples illustrating the invention will be described hereinafter
in a more detailed manner and by reference to the embodiments represented
in the Figures.

[0200] To determine the effectiveness of compositions of the invention in
promoting wound healing and/or bone and/or tissue regeneration, the
following experiments are performed.

[0201] Whole human blood samples are collected in a separator tube
according to the invention. A separator tube according to the invention
is for example an approximately 15 mL glass tube (16 mm diameter and 130
mm in length) containing 3 mL of polyester-based thixotropic gel as well
as 1 mL of sodium citrate solution at 0.1 M and containing a usable
vacuum of or about 8.5 mL. This separator tube constitutes a ready-to-use
device for the preparation of a platelet concentrate composition of the
invention (also called RegenTHT® (Thrombocyte Harvesting Tube) from
Regen Lab, Switzerland).

[0202] Another example of a separator tube according to the invention is a
tube of approximately 10 mL in PET (polyethylene terephthalate)
containing 1 mL of a thixotropic gel comprising a polymer mixture and
anhydrous sodium citrate deposited on the inner surface of the tube by
spraying (about 3.5 mg per mL of blood) and containing an usable vacuum
of or about 8 mL, constitutes a ready-to-use device for the preparation
of a platelet concentrate according to the invention (also called
RegenBCT® (Blood Cell Therapy) from Regen Lab, Switzerland).

[0203] These tubes are sterilized by irradiation (such as prescribed by
ISO 11137, UNI EN ISO 11737-2, UNI EN 552, UNI EN 556) and hermetically
sealed by a traditional cap such mottled bromobutyl conventional rubber
stopper for the glass tube and a chlorobutyle stopper having a
polyethylene cover for the operator safety.

[0204] Then, the separator tube is centrifuged at or about 1,500 g up to
or about 2,000 g for about 3 to 10 min, i.e. of or about 2,500 rpm up to
or about 3,800 rpm with a centrifuge with a swinging rotor, having a
radius of 20 cm. In case of a centrifuge having a rotor with a fixed
angle of about 45°, the centrifugation time should last for at
least about 15 min.

[0206] For the therapeutic applications, a kit according to the invention
adapted for tissue regeneration is used wherein the kit (also called
RegenKit®) comprises two sterile blisters comprising:

[0207] one
blister (RegenPRP®) comprising accessories for the phlebothomy,
separator tubes of the invention (RegenTHT® or RegenBCT®),
optionally vials of ETOH and CaCl2 for the preparation of an
autologous thrombin serum (RegenATS®).

[0208] one blister
(RegenApplicator®) comprising two syringes (e.g. 1 mL and 1 or 3 mL),
holders and clamper and a tip applicator with a dual exit.

[0209] For the preparation of cell compositions, according to the
invention, the cells are prepared according to the general protocol as
follows:

a) Biopsy

[0210] A biopsy of the corresponding tissue is obtained under sterile
conditions using standards methods adapted to the specific cell that will
be collected. The cells are used extemporaneously or optionally after
ex-vivo culture and cell proliferation as follow.

b) Ex-Vivo Culture and Cell Proliferation

[0211] Cells used for the preparation of cell compositions according to
the invention, such as keratinocytes, bone marrow cells, fibroblasts;
periosteum or corneal cells, such as corneal limbal stem cells;
melanocytes and Langerhans cell; fat cells; muscle cells such as
myoblasts and satellite cells; osteoblasts; chondrocytes; umbilical cord
cells; Schwann cells, tendon or pancreatic cells are expanded in a cell
carrier medium (e.g. DMEM or Ham's) on plates (e.g. Petri dishes or
culture flask) coated with a platelet concentrate according to the
invention, preferably autologous, enriched with fibronectin. The culture
media may be enriched preferably with DMEM for example in the case of
keratinocytes. For cells such as bone osteoblasts, chondrocytes and
myoblasts, enzymatic digestion of the corresponding tissue in presence of
for example collagenase or trypsin is necessary before plating.
Incubation on the plates is performed at 37° C. under a gas flow
of 95% oxygen or air and 5% carbon dioxide. Typically, incubation time
vary from 10 to 20 min. The cell expansion may be increased (like for
example in the case of myoblast, fibroblast and chondrocyte cells) by
phototherapy (e.g. light exposure at 633 nm of about 10 min at 2
J/cm2, once a week during the incubation phase).

[0212] The explants may be cultured in Petri dishes or culture flask using
air-lifting technique (Molnar et al., 1996, Tissue & Cell, 28:547-556)
and air interface method (Meller et al., 2002, Br. J. Opht., 86, 463-471)
with half of the explant exposed to air. The culture medium is changed
regularly during incubation, such as every 3 days. The expansion of the
cells in a 2D mode as planar monolayers, is obtained for example for
myoblast, fibroblast and chondrocyte cells. A 3D cell growing pattern can
be obtained for example for corneal, myoblast, fibroblast, chondrocyte,
adipocyte and keratinocyte cells, by adding diluted autologous platelet
concentrate composition according to the invention at about 5 to about
40% volume of plasma or enriched plasma to the culture medium. Typically,
the addition of diluted autologous platelet concentrate composition
according to the invention is performed 2 or three times during the
incubation time. The 3D biological scaffold then obtained allows to
enhance the extra-cellular matrix which is useful for autologous stem
cell transfer.

[0213] After incubation, cells are then released from dishes with gentle
trypsin digestion that lifts off the cells and allows them to be
pelleted.

c) Cell Quality and Safety Check

[0214] The cell viability in the so-obtained cell preparation is checked
by microscopic cell count, flow-cytometer cell count together with
immunochemistry on tissue markers by standard techniques. Cell viability
is also tested via trypan-blue just after cell release by trypsin. Safety
of the preparation is also checked through contamination check via
microbiology assay to exclude contamination with viruses or bacteria and
to avoid transfer of zoonotic infections. The use of FCS is avoided thus
preventing transmission of Mad Cow Disease.

d) Administration of the Cell Preparation

[0215] The cell preparation obtained above is placed in autologous
platelet concentrate composition according to the invention as cell
carrier vehicle for transport before delivery to the patient. Then, the
cell preparation obtained above is injected or transplanted into the
patient. The injection or transplantation mode has to be adapted to the
type of cells contained in the cell preparation according to the
invention and to the aimed therapeutic or aesthetic effect. More details
are given in the Examples below on the method for the preparation and use
of the cell compositions according to the invention more specifically,
depending on the type of cells and aimed therapeutic or aesthetic effect.

[0216] Keratinocyte cell or fibroblast cell preparations according to the
invention may be used readily after collection or after cell culture as
described above. However, the cell preparations according to the
invention are preferably prepared after cell culture as described above.

[0217] The cell preparations according to the invention present a better
viability and stability (including integrity of cell properties preserved
such as ability the synthesize proteins and deliver growths factors) than
cells prepared in a medium without autologous platelet concentrate
composition according to the invention. Further, cell proliferation so
obtained is enhanced: cells grow faster (about 3 to 5 days quicker) and
are denser compared to control mediums and serum starved media. The
advantage of the process for the preparation of a cell composition
according to the invention is that the same autologous medium is used as
vector for cell culture, cell preservation, cell injection, vector for
cell bio-stimulation and tissue regeneration.

Example 1

Preparation of an Autologous Platelet Concentrate

[0218] Separator tubes of the invention are beforehand tested for the good
tolerability, the non-toxicity and the non-mutagenicity of the
thixotropic gel according to norms ISO 10993-11, ISO 10993-10, ISO
10993-12 and ISO 10993-3.

[0219] About 8.5 to about 10-mL of human blood sample are collected within
the separator tube of the invention, where the blood is aspirated by the
vacuum. The mixture is then centrifuged at approximately 3,800 rpm for
about 3.5 min. The platelet-rich plasma is then collected.

[0220] The analysis of the platelet concentrate obtained by the method of
the invention has shown that it contains 2 to 4 times the normal levels
of platelets and growth factors, compared to a natural blood clot, while
maintaining normal levels of fibrin and fibrinogen and containing
practically no blood cells (<1% hematocrit, compared to 35-50% in a
normal blood clot and 15-20% in platelet-rich plasma obtained from known
methods of preparation). The study shows also the presence of leukocytes
glycoprotein fibronection and this demonstrates that the coagulating
properties are preserved.

[0221] The composition of the platelet concentrate (also called
RegenPRP® from Regen Lab, Switzerland) compared to whole blood, whole
plasma and platelet-poor plasma is presented in table 1 below:

[0223] The platelet yield obtained by such the method of the invention has
been measured (90-99%) and shows to be drastically increased in
comparison with the platelet yields (30-62%) obtained from known methods
of preparation described in Marx et al., 2004, above.

[0224] In addition, it has been shown through ELISA kits (R&D Systems,
Inc.) and the response to coagulation activation of the platelet
concentrate of the invention, that the activity of coagulation factors is
preserved: the concentration of D-dimers (fibrin breakdown products),
known markers of coagulation activation, and the lysis process are stable
and therefore the coagulation properties of the platelet concentrate are
not weakened by the process of the invention.

[0225] The levels of growth factors (PDGF, EGF, TGF-β and VEGF) from
the platelet concentrate of the invention are demonstrably stable for a
period of at least 72 hours (4 days) when stored at room temperature in
the sterile separator tube of the invention. The evolution of growth
factors PDGF BB, EGF and VEGF over 72 hours is presented on FIG. 1.

[0226] The properties of the platelet concentrate according to the
invention make it possible to envisage preparing platelet concentrate
obtained using the invention's procedure, one to several days before a
reparative surgery, in order to reduce the workload in the operating room
and speed-up the surgical procedure.

[0227] For subsequent therapeutic use, the autologous platelet concentrate
is generally mixed with a conventional coagulation activator such as a
thrombin activator (e.g. calcium chloride pour example at 10%),
optionally mixed with a fibrinogen activator such as thrombin, preferably
homologous (e.g. 10 UI to 100 IU per mL of plasma), batroxobin (e.g. 20
BU per mL of plasma) or a thrombin enriched preparation.

Example 2

Therapeutic Use of the Autologous Platelet Concentrate of the Invention

[0229] One 88-year-old patient (Patient 1) suffering from multiple
locations Kaposi's angiosarcoma on lower limbs and from a radio-induced
necrosis on the left leg. The radio-induced necrosis was resulting from
radiotherapy treatment. After 12 months after the end of the low-voltage
X-ray treatment, the necrosis was consisting in a deep superinfected
ulcer surrounded by a scab (35×25 mm). The wound had been
previously unsuccessfully treated with various treatments such as with
steroids and healing creams.

[0230] One 81-year-old patient (Patient 2)
suffering from a vertex spinocellular carcinoma was presenting a
cutaneous ulceration (about 10 mmm diameter) with peripheral dyskeratosis
without any infection sign resulting from a biopsy-resection and a
post-surgical radiotherapy (total dose of 52 Gy).

[0231] One 60-year-old
patient (Patient 3) having received a pre-surgical irradiation (7 Gy) for
tibia and fibula synostosis on the right leg was presenting a
radio-induced necrosis consisting in a deep ulcer (50×30 mm
diameter) without inflammation.

b) Treatment:

[0232] 8.5 mL of blood sample is taken from each patient and centrifuged
in a separator tube as described in Example 1, according to the protocol
as described in Example 2. The resulting platelet concentrates are then
mixed with calcium chloride at 10% vol. Each autologous platelet
concentrate composition is then applied on the radio epidermitis wound
site of the corresponding patient. The wound is then covered and
protected with humid compresses (Day 1).

[0233] Between days 3 and 5, the wound status is checked and the wound
dressing is changed. At day 7±1, a new application of a new autologous
platelet concentrate preparation of the invention is performed. If
needed, the same treatment sequence is followed with the same time
intervals till the complete cicatrisation of the wound.

[0237] These results show the benefit effect of the platelet concentrate
composition of the invention in the healing of chronic radio-induced
ulcers, even in the case of those which were resistant to previous topic
treatments and in the absence of any allergic reaction.

Example 3

Therapeutic Use of the Autologous Platelet Concentrate of the Invention in
Combination with an Autologous Thrombin Enriched Serum

[0238] To activate coagulation, an alternative to the mixture of the
platelet concentrate of the invention with a thrombin activator before
the use on a patient, as described in Example 1, is the combination of
the platelet concentrate of the invention with a fibrinogen activator
such as a thrombin enriched composition and preferably with a thrombin
serum (e.g. autologous) according to the invention.

a) Preparation of an Autologous Thrombin Serum (ATS)

[0239] An autologous thrombin serum to be used as a thrombin enriched
preparation in the context of the invention is prepared by a process
which comprises the addition to a patient's whole blood sample (e.g. 8
mL) collected in a separator tube of the invention as described in
Example 1, a 95% v. ethanol solution (e.g. 1 mL) and calcium chloride 10%
(e.g. 1 mL). The mixture (also called RegenATS® from Regen Lab,
Switzerland) is then allowed to precipitate for about 30 min at room
temperature.

[0240] After 30 min, almost 80% of the anti-thrombin (among other proteins
like fibrinogen) is precipitated; then the tube is centrifuged at or
about 1'500 g for about 8 to 10 min and the autologous thrombin serum is
ready for use in combination with the platelet-rich concentrate of the
invention.

b) Combined Preparations

[0241] One of the originality of this process is that after the initial
step of incubation of the autologous thrombin serum preparation process
(e.g. at least about 30 min), the separator tubes of the invention
containing respectively the autologous thrombin serum preparation and the
platelet concentrate preparation can be centrifuged simultaneously in
order to get the two blood extract preparations ready for use at the same
time.

c) Combined Use

[0242] To allow the polymerization of fibrinogen into a fibrin mesh (which
occurs during the coagulation process) to occur only at the moment of
application of the platelet-rich preparation on the wound, the platelet
concentrate composition and autologous thrombin serum (coagulation
activator) are applied simultaneously at a vol. ratio of about 10:1 to
about 10:3 (concentrate to coagulation activator ratio) to the wound.

[0243] The simultaneous delivery of both preparations is achieved for
example by a device comprising two syringes (e.g. 10-mL syringe for the
platelet concentrate composition and a 1-mL or 3 mL syringe for the
thrombin serum), that releases the preparations simultaneously so that
they mix and polymerize upon contact with the wound.

Example 4

Therapeutic Use of the Autologous Platelet Concentrate of the Invention in
Combination with Skin Cell Extract

[0244] A total of 35 patients having received a skin graft (representing
less than 15% of the skin surface) have been included in the study.
Patients treated with immunosuppressants or corticoids or with renal
insufficiency or severe peripheral artheropathy were excluded.

[0245] All the following manipulations are performed under the strict
rules of asepsy and sterility.

Group A: 13 Patients

a) Preparation of Platelet Concentrate

[0246] A 8.5 mL sample of whole blood from each patient (from a higher
limb where no perfusion is present) is collected in a separator tube
according to the invention. The separator tube with the whole blood is
immediately centrifuged during about 8 min at 2,800 rpm. Before the
enriched plasma (PRP) is collected, the operator discards the half or 2
mL of the supernatant and then re-suspends the platelets in the remaining
plasma. The platelet-rich concentrate is then transferred to a sterile
tube maintained at a temperature of 37° C.

b) Wound Coating

[0247] The autologous platelet concentrate of the invention (also called
RegenPRP®) is mixed with a solution of calcium chloride 10% in a ratio
10:1 and the graft donor site (where skin was removed) of each
corresponding patient is coated with the autologous corresponding mixture
in order to obtain coagulation of the platelet concentrate on the wound.

Group B: 8 Patients

a) Skin Cell Sampling on the Patient

[0248] Keratinocytes are extracted from each of the patients from this
group. A thin healthy skin sample (about 2 cm2) is removed from each
patient and washed three times in a PBS solution. The washed biopsy is
then deposited in a Petri dish containing trypsin and cut into very small
fragments (0.5 cm*0.5 cm) with a scalpel. The skin fragments are then
incubated during 45 min at 37° C. on a stirring device in 20%
volume of autologous platelet concentrate composition according to the
invention, also called RegenPRP®, obtained above. The supernatant is
then collected, centrifuged and cells are re-suspended in a PBS solution.
The keratinocytes count is determined under microscope. Finally, the
obtained keratinocytes were re-suspended in the autologous platelet
concentrate according to the invention (5-40% vol.) from the
corresponding patient.

b) Preparation of Platelet Concentrate

[0249] The procedure is the same as for Group A.

c) Wound Coating

[0250] The keratinocyte suspension (also called RegenExtracell®) is
applied as soon as ready (the entire preparation not exceeding a day) on
the wound on the same way as described in the case of Group A.

Control Group: 14 Patients

[0251] The graft donor site of each patient of this group is coated with a
non-therapeutic compress (Jelonet®).

Randomization and Treatment

[0252] In the surgery bloc, after the graft skin removal, the donor site
is coated with a temporary compress soaked with an adrenaline solution (1
ampoule of 1 mg/mL of adrenaline diluted in 500 mL NaCl 0.9%) and
depending on the randomization table, the donor site is treated according
to the three following methods:

[0253] Groups 1 and 2: Coating of the wound with the respective wound
healing composition and covering of the wound with a non-therapeutic
compress (Jelonet®).

[0254] Group 3: Direct covering of the wound with a non-therapeutic
compress (Jelonet®).

[0255] The compresses are then covered with Kerlix® bands and elastic
bands such as "Velpeau".

Treatment Efficacy Criteria

[0256] The efficacy of the treatment is evaluated according to 3 criteria:

[0257] The time needed for the complete cicatrisation of the treated site
(healing time or HT in days)

[0258] The epithelization (evolution of the cicatrisation progress)
measured at day 5 after the treatment according to 7 degrees:

[0259] 0: Absent

[0260] 1: Slight

[0261] 2: Moderate

[0262] 3: Important

[0263] 4-7: Very important, increasing degrees of importance;

[0264] The pain evaluated at day 5 after the treatment by the patient
him/herself, generally at the time of compress change on a scale from 0
to 10 (0: no pain and 10: extreme pain).

[0265] The compress is opened at day 5 post-surgery to allow the
evaluation of the quality of the treatment and covered with new
Jelonet® compresses covered with dry compresses.

[0266] The compress is then changed very two days till the complete
cicatrisation. Any side effects or medical complications are watched
during the whole duration of the cicatrisation process.

Results

[0267] The results of the treatments for each patient group (Control
group: C, Group A: RegenPRP®, Group B: RegenExtracell®) are
presented on FIG. 2 in terms of healing time in days (HT), pain at day 5
(P) and epitalization at day 5 (E). The dotted line indicates when the
first bandage is changed at day 5.

[0268] The cicatrisation process is clearly stimulated by the use of the
platelet concentrate of the invention as compared to the control group.
The quality of the cicatrisation is also better in the case of the use of
platelet concentrate of the invention. In addition, the pain at the donor
site is dramatically reduced in the case where the platelet concentrate
of the invention was used as compared to the control group.

[0269] All the beneficial effects of the platelet concentrate of the
invention are increased when a mixture of keratinocytes suspended in the
platelet concentrate of the invention is used.

[0270] The mean healing time is of 7 days for the group treated with a
platelet concentrate of the invention and 5 days when keratinocytes are
suspended in the platelet concentrate as compared to an average of 12
days in the control group.

[0271] Tolerability was excellent and no side effect or allergy has been
detected.

[0272] This shows that the platelet concentrate of the invention alone or
combined with keratinocytes is very efficient in accelerating the wound
healing process and not only decreases the pain, but also the
inflammatory reaction and improves the final aspect of the scar.

[0273] Alternatively, using the same process of dissociation, skin cells
can be placed in a Petri dish coated with the autologous platelet
concentrate composition according to the invention, also called
RegenPRP®, obtained above and cultured for 2 to 5 days. Then, before
the graft, the obtained skin cell preparation may be sprayed onto the
wound, in order to prepare the site for a better bio integration of the
implanted cells, and a better expansion in vivo.

Example 5

Cosmetic Use of the Autologous Platelet Concentrate of the Invention

[0274] An autologous platelet concentrate composition is prepared as
described in Example 1.5 mL of this platelet concentrate composition
(also called RegenACR®: (Autologous Cell Rejuvenation) from RegenLab,
Switzerland) is injected subcutaneously in a wrinkle groove as wrinkle
filling material, in the same way as commonly done with other wrinkle
filler such as hyaluronic acid. The deepness of the wrinkle is
progressively decreasing within the first weeks after the treatment and
at the site of injection, a very clear regeneration of the area is
obtained with an optimal result at two to three months. As opposed to
what observed with other wrinkle filling materials, neither inflammation,
nor swelling is observed at the site of injection and the benefit is
durable as opposed to hyaluronic acid which is bio-resorbed after 4 to 6
months.

[0275] Known methods to study the effect autologous platelet concentrate
compositions of the invention on wrinkle deepness can be used to such as
a three-dimensional reconstitution of skin relief by optical profilometry
(stylus method) (Grove et al., 1989, J. Am. Acad. Dermatol., 21: 631-7)
or by laser microscopy on silicon skin replicas. Another method consists
in the in vivo quantification of the skin surface "Surface evaluation of
living skin" or "SELS" through the analysis of images in UV light
(Tronnier et al., 1997, Akt. Dermatol., 23:290-295). Another method for
the surface evaluation of living skin is based on an optical system with
a CCD camera measuring the four skin parameters: roughness, scaling,
smoothing and wrinkling (Fluhr et al., 1995, Akt. Dermatol., 21:151-156).
Dep dermal augmentation can be assessed by ultrasound, Dermascan®,
Denmark).

[0276] Other examples of cosmetic use of the autologous platelet
concentrate of the present invention include:

[0277] Admixing the platelet concentrate according to the invention with a
cream, preferably an emulsion, before application to a wound, after
surgery or on healthy skin. During the absorption process, the platelet
preparation is carried into the skin by the cream or emulsion in order to
amplify the hydrating benefit and to bio-stimulate the regeneration or
rejuvenation of the skin.

[0278] Using a hydrogel like the Albugel (EP 1 543 846) preparation of
100% Albumin or any other hydrogel resulting from the reticulation of
Albumin and other chemical compound like polyethylene glycol or any other
ingredient, using a paper based highly hydrophilic, a carrier to leave in
contact with the skin until the platelet rich plasma is absorbed.

Example 6

Autologous Muscle Cell Association Preparation

[0279] Example of autologous cell association according to the invention
can be prepared by using the process according to the invention wherein
skeletal muscle cells (muscle progenitor cells or satellite stem cells)
are provided under step (d) or (e).

a) Myoblast Progenitor Stem Cells

[0280] Skeletal muscle biopsy is obtained from the Vastus lateralis and
measures 7×3 cm. Muscle is primed the day before biopsy, with
intra-muscular injection at proposed biopsy site (10 by 15 cm skin area
on lateral aspect of thigh overlying the vastus lateralis muscle and just
above the knee joint, on either side) with Decadon and Marcaine (long
acting Lignocaine) Muscle is diced and enzymatically digested with
combination of collagenase, pronase and trypsin (Worthington). Enzyme
action is neutralised using patients serum in DMEM culture medium. Muscle
explants are plated Petri dishes coated with the autologous platelet
concentrate composition according to the invention, also called
RegenPRP® (prepared as described in Example 4) and incubated in 95%
oxygen and 5% carbon dioxide at 37° C. for 3 to 4 weeks. Desmin or
CD-56 expression is used as myoblast marker to identify myoblasts from
fibroblasts. Myoblast progenitor cell proliferation in 3D is shown on
FIG. 3.

[0281] Cell proliferation can be enhanced by photo-light exposure at 633
nm of 2 J/square centimetre for 10 min during culture. The day of
transplantation (e.g. after 3 to 4 weeks of incubation), the skeletal
muscle cells are released are released by trysin and placed in the
autologous platelet concentrate composition according to the invention,
also called RegenPRP® (prepared as described in Example 4). Injections
into the myocardium can be made as direct injection or multiple catheter
injections into the left ventricle myocardium. The myoblast cell
preparation according to the invention is useful for cardiac disorders
such as heart regeneration, treatment of heart failure, chronic cardiac
failure, ischemic and non-ischemic cardiac failure and non-ischemic
cardiomyopathy. Ejection fraction can be improved by 9% for cardiac
recipients of skeletal myoblasts.

[0282] The above cell preparation may also be useful for in the treatment
of urinary incontinence (myoblast cell extracts prepared as described
above and injected into the bladder neck), reflux oesophagitis or
gastro-oesophageal reflux disorder (myoblast cell extracts prepared as
described above injected into the lower oesophageal sphincter) and anal
incontinence (myoblast cell extracts prepared as described above and
injected in para-anal area).

[0283] Alternatively, a combined preparation of fibroblast and myoblast
may be carried out (fibroblasts are present in the muscle biopsy and
sprout from the perimysium along side the myotubes and satellite stem
cells).

[0284] In case of the treatment of cardiac disorders, a mix of fibroblast
cell preparation and myoblast cell preparation (obtained as indicated
above) is inserted into the myocardium in a ratio fibroblast/myoblast of
about 30:70.

[0285] For bladder neck incontinence treatment, a separate culture of
fibroblasts is made at the same time as the myoblasts as described above
and the fibroblast cell preparation is injected para-urethrally and
myoblast cell preparation is injected into the rhabdosphincter, under
ultrasound control.

b) Satellite Stem Cells

[0286] Myoblasts and satellite stem cells are cultured ex vivi in presence
of autologous platelet concentrate composition according to the
invention, also called RegenPRP®. Cell proliferation priming is
observed after 7-days of primary culture.

[0287] Cells are then harvested after incubation of about 3-4 weeks and
placed in tissue culture medium (DMEM plus 5-10% vol. autologous platelet
concentrate composition according to the invention) containing a human
de-epitheliased amnion patch measuring 4×4 cm and the autologous
platelet concentrate composition according to the invention, also called
RegenPRP® (prepared as described in Example 4). The preparation is
then subjected to UV irradiation for 10 min. During incubation (typically
about 2 to about 3 weeks), the cells spread over the amnion construct and
form a monolayer. Viability and monolayer progress is assessed by twice
weekly biopsy of patch edge and histological assessment for thickness of
monolayer.

[0288] The day of transplantation (e.g. after bout 3 to 4 weeks of
incubation), the ventricular surface is spread with the autologous
platelet concentrate composition according to the invention, also called
RegenPRP® (prepared as described in Example 4) and then the patch
obtained above is placed with cells down side onto a raw surface of the
ischemic ventricle in order to allow the stem cells on the patch to
populate the ischemic segment after ventricular injection. Cell retention
is maintained by the amnion that is inert and induces no immunological
reaction.

[0289] The satellite stem cell preparation according to the invention is
useful for heart regeneration and treatment of heart failure as tissue
engineering preparation for cardio myoplasty.

Example 7

Autologous Fibroblast Cell Association Preparation

[0290] Example of autologous fibroblast cell association according to the
invention can be prepared by using the process according to the invention
wherein dermal fibroblast cells are provided under step (d) or (e).

[0291] Dermal fibroblasts are isolated and expanded according to the
following procedure:

[0292] One month before biopsy, the prime donor skin area (behind an ear
of anterior axillary fold, e.g. non solar aged area) is treated with
vitamin A cream to activate the dermal fibroblasts. A skin biopsy of
10×6 mm full thickness is performed and dissected under microscope
to remove all epithelium. The-epithelialize biopsy (dermis) is then cut
into 3×3 mm blocks as explants. The papillary dermis is then placed
upwards and cultured using air-lifting technique (Molnar et al., 1996,
above) and air interface (Meller et al., 2002, above) with half of the
explant exposed to air. The explants are plated (e.g. 6 explants per
well) in DMEM and cultured at 37° C. at 95% oxygen and 5% CO2
for about 3-5 up to about 9 days in Petri dishes or culture flask. The
medium is changed every 3 day. The fibroblasts expansion in 2D mode as
planar monolayers, as static growth is observed during incubation. At
days 7 to 9 after the start of incubation, a change in proliferation and
phenotype pattern to 3D is obtained by adding diluted 5-10% autologous
platelet concentrate composition according to the invention, also called
RegenPRP® (prepared as described in Example 4) to the culture medium:
cells are primed with RegenPRP® (0.2 ml per well) just to cover base.
Cells grow then as a 3D fibrin gel matrix (FIG. 3). Cells then
differentiate to form biological scaffold or network in fibrin gel such
as shown on FIGS. 4a and 4b. Cell number is measured by daily counting
under a grid and to assess apoptosis: use inverted microscope
(Olympus®).

[0293] After 3 to 6 weeks of incubation, the cells are harvested from the
fibrin gel. Cell viability is assayed with classical Trypan blue method
and with bacteriological evaluation, including virus contamination.

[0294] The expanded fibroblast cell extract obtained above is placed in a
syringe in presence of autologous platelet concentrate composition
according to the invention, also called RegenPRP® and the preparation
is injected into face wrinkles, more specifically under the wrinkles
Injections must be performed over the whole face to cover forehead,
jowls, molar region, cheeks, chin and neck.

[0295] Cell expansion may be increased by photo light exposure of cell
culture at 633 nm. The fibroblast cell preparation according to the
invention is useful for facial rejuvenation, amelioration of facial
wrinkles and rhytids, treatment of skins damaged by radiations
(radiodermatitis or sun damaged skin), aged skins or burned skins and/or
in the amelioration of facial wrinkles, rhytids, acne (especially after
dermabrasion treatment), burns, rubella or small pox scars, vitiligo,
lipoatrophy or lypodystrophy, such as AIDS-related lypodystrophy;
Kaposi's sarcoma, skin skeloids or Dupuytren's palmar fibromatosis and/or
in skin rejuvenation treatments.

Example 8

Autologous Fat Cell Association Preparation

[0296] Example of autologous cell association according to the invention
can be prepared by using the process according to the invention wherein
adipose stem cells are provided under step (d) or (e).

[0297] Adult adipose stem cells are isolated by standard culture method in
5-10% vol. an autologous platelet concentrate composition according to
the invention, also called RegenPRP®. The preparation is then injected
with an applicator into patients suffering from tissue deficiencies, such
as post traumatic deficiencies or aged-related deficiencies for patients
being around about 40 years-old.

[0298] The fat cell preparation according to the invention is useful for
the treatment of lipoatrophy such as in HIV/AIDS patients and others
congenital hemiatrophy of the face.

Example 9

Autologous Chondrocyte Cell Association Preparation

[0299] Example of autologous cell association according to the invention
can be prepared by using the process according to the invention wherein
chondrocyte cells are provided under step (d) or (e).

[0300] Cartilage is isolated from the donor's knee (biopsy size 10×5
mm) and diced. The cartilage chondrocyte cells are cultured for 4-6 weeks
in medium enriched with an autologous platelet concentrate composition
according to the invention, also called RegenPRP®. Cartilage cells are
then released by enzymatic digestion (collagenase and pronase). The cell
preparation is then incorporated surgically into the patient with deep
chondral defects and damage.

[0301] The chondrocyte cell preparation according to the invention is
useful for the treatment of deep cartilage damage and erosion or
arthroscopy.

[0302] Another example of the use of a chondrocyte cell preparation
according to the invention is the use in rhynoplasty without surgery by a
single injection procedure: A patient suffering from congenital cartilage
nose atrophy.

[0303] The day before injection, a biopsy of the cartilage of the hear
0.4*0.4 cm is performed and placed in a sterile recipient filled with
DMEM and antibiotic. The biopsy is treated with enzymatic digestion
including thrypsin and collagenase. The released chondrocytes are then
re-suspended in the autologous platelet concentrate composition according
to the invention where 10% CaCl2 have been added.

[0304] The patient receives first a local anesthesia, and nose
disinfection. Then, the above chondrocyte preparation is injected on the
cartilage surface and or periosteum membrane of the site requiring
augmentation of volume or lift. In a second phase, autologous platelet
concentrate composition according to the invention where 10% CaCl2
have been added is injected into the superficial part of the nose skin,
in order to biostimulate regeneration and the rejuvenation of the skin.
After one hour, the injection is achieved and the patient could return
home. An exceptional recovery of viable cells is observed: the amount of
chondrocyte cells and plasma cells recovered and injected was about
109 cells.

[0305] The chondrocyte cell preparation according to the invention is
therefore useful for the treatment of nasal cartilage defects, without
surgical procedure, but only by injection.

Example 10

Autologous Umbilical Cord Stem Cell Association Preparation

[0306] Example of autologous cell association according to the invention
can be prepared by using the process according to the invention wherein
umbilical cord stem cells are provided under step (d) or (e). Umbilical
cord stem cells are isolated and then cryo-preserved and used to treat
blood disorders.

[0307] The umbilical cord stem cell preparation according to the invention
is useful for the treatment of haematological diseases (like
Thalassaemia).

Example 11

Autologous Tendon Cell Association Preparation

[0308] Example of autologous cell association according to the invention
can be prepared by using the process according to the invention wherein
tendon cells are provided under step (d) or (e).

[0309] Tendon fibroblast cells are isolated according to procedure
standard procedures in 5-10% vol. of autologous platelet concentrate
composition according to the invention. The tendon fibroblast cells are
cultured for about 1 to about 3 weeks in culture medium enriched with an
autologous platelet concentrate composition according to the invention,
also called RegenPRP®. The cell preparation is then injected into the
patient at the injury site (e.g. tendon torn, arthritic area). The
injection can be guided by echographie, for localisation of the damaged
site, and better graft of the implanted solution.

[0310] The injection of the tendon fibroblast cell preparation may also be
performed next to rotator cuff in should: first the rotator cuff tear is
repaired arthrosopically, then the tendon fibroblast cell preparation is
injected via a long catheter onto the sutured area. This improves the
healing of the tendon fibroblast at the edge of the rotator cuff, prevent
haematoma in confined space under the acromion, prevents frozen shoulder
by speeding up healing and enhancing rehabilitation and joint movement.

[0311] The tendon cell preparation according to the invention is useful
for the treatment of tendons torn, arthritis in joint caused by traumas
or by aging, rotator cuff in shoulder.

Example 12

Autologous Ligament and Gingival Cell Association Preparation

[0312] Example of autologous cell association according to the invention
can be prepared by using the process according to the invention wherein
periosteal membrane and gingival cells are provided under step (d) or
(e).

[0313] Under general and local anesthesia, periosteum (approximately
10×10 mm) is aseptically harvested from the buccal side of the
mandibular body in four healthy female beagle dogs. The harvested
periosteum is cut into 3×3 mm pieces. The tissues are placed
directly on a 6-well plate and cultured (for about 3 to about 6 weeks) in
a humidified atmosphere of 5% CO2 and 95% air at 37° C. in a
culture medium enriched with an autologous platelet concentrate
composition according to the invention, also called RegenPRP®. The
periosteal membrane and gingival cells are isolated by enzymatic
digestion and cultured by static technique.

[0315] Patients are separated into two groups: (1) a control group which
receives an autologous platelet concentrate composition according to the
invention without periosteal membrane cells and (2) a treatment group
which receives the periosteum membrane cell preparation obtained above.
The autologous platelet concentrate composition (in control group) and
the cell preparation (for the treated group) are respectively injected
into the patient at the injury site.

[0316] 12 weeks after operation, the periosteal membrane cell preparation
had completely disappeared in both control and treatment group. Bone
regeneration in the treatment group with cultured periosteum membrane was
significantly greater than that in the control group: the thread of
dental implants was almost covered with the regenerated bone, while most
of the thread in the control group was exposed. The treatment group
demonstrated relatively thick and dense lamellar bone formation from
bottom to top of the created defect. Thick layers of woven bone were
attached to the implant surface and osteoblast like cells were observed
around the surface. Although abundant neovascularization was observed in
the bone matrix, inflammatory cells were rarely observed. The border
between the regenerated and original bone was not clear. In the control
group, specimen exhibited thin cortical bone formation at the dehiscence
defect. Scarce woven bone was observed between the implant surface and
the cortical bone, and few osteocytes and osteoclasts were observed
within the bone matrix and at the surface, respectively. Bone density was
significantly higher in the treatment group than the control group. The
mean LF values were 77.58±1.14% and 37.03±4.63% in the treatment
and control groups, respectively (P<0.05).

[0317] The periosteal membrane and gingival cell preparation according to
the invention is useful for the treatment of periodontal disease and dry
sockets, especially in promoting bone regeneration at sites of implant
dehiscence.

Example 13

Autologous Corneal Cell Association Preparation

[0318] Example of autologous cell association according to the invention
can be prepared by using the process according to the invention wherein
corneal cells are provided under step (d) or (e).

[0319] A biopsy is taken from the epicanthis on the edge of the cornea and
the corneal limbal stem cells were expanded for autologous
transplantation in the same person after 4 weeks of culture in
Petri-dishes or flasks coated with an autologous platelet concentrate
composition according to the invention, also called RegenPRP®.

[0320] The corneal corneal cultured stem cells (of limbal origin) may be
ex vivo engineered onto the surface of de-epithelialised human amnion in
a monolayer, after seeding the construct with a suspension of cultured
and viable corneal keratinocytes according to the invention. About
500,000 cells are used for the seeding and the cells are allowed to cover
the surface of the construct with cells after further incubation of about
another 3 weeks. The engineering with cells occurs after about three
weeks of primary cell culture, and re-seeding may be necessary. The
resulting biological cell-biocomposite construct consisting of collagen,
amnion fibers and corneal keratinocytes, consisting of membrane and
monolayer of cells.

[0321] The corneal cell preparation according to the invention can be
spread onto a dissolvable contact lens that is applied to the damaged
cornea. The contact lens disappears and the cells close the corneal
defect.

[0322] The corneal cell preparation according to the invention can be
administered topically in eye drops in patients suffering from dry eye
symptoms. Alternatively, the above amnion can be used on its own on the
scarred cornea or the construct and the cell preparation according to the
invention can be attached to the inside of a biological or artificial
contact lens and then applied to the cornea and covered with a eye pad.

[0323] The corneal cell preparation according to the invention is useful
in alleviating the pain of dry eye, for the treatment of Steven's Johnson
Syndrome and corneal blindness due to acid and corrosive alkali burns in
industry, corneal ulcers such as recalcitrant neurotrophic, herpetic and
immunologically induced corneal ulceration.

Example 14

Autologous Bone Marrow Cell Association Preparation

[0324] Example of autologous cell association according to the invention
can be prepared by using the process according to the invention wherein
bone marrow cells are provided under step (d) or (e).

[0325] Hip bone marrow is harvested and centrifuged in a ready-to-use
device for the preparation of a platelet concentrate according to the
invention (also called RegenBCT® (Blood Cell Therapy) of in order to
separate red blood cells.

[0326] The bone marrow cell preparation is then admixed to the platelet
concentrate according to the invention and applied or injected with an
applicator with addition of CaCl2 to the injured site of the
patients.

[0327] The bone marrow cell preparation according to the invention is
useful for the treatment of ischemic and non-ischemic cardiac disease,
bone defect, cartilage defect.

Example 15

Autologous Schwann Cell Association Preparation

[0328] Example of autologous cell association according to the invention
can be prepared by using the process according to the invention wherein
Schwann cells are provided under step (d) or (e).

[0329] Under local anaesthesia, a biopsy is performed either the N.
Saphenous of N. SURALIS in the lower extremity. The nerve biopsy is cut
into small blocks and primary cultures are induced in Petri dishes
enriched an autologous platelet concentrate composition according to the
invention, also called RegenPRP®.

[0330] Monolayers are expanded in 3D and the cells are eventually
harvested by trypsin digestion and concentrated in a syringe for local
infiltration of the surgically exposed and damaged spinal cord. The
cultivated cells have been shown to contain myelin.

[0331] The Schwann cell preparation according to the invention is useful
for the treatment of peripheral nerve damage, nerve suture and spinal
cord injury.

Example 16

Autologous Human Islet Cell Preparation

[0332] Example of autologous cell association according to the invention
can be prepared by using the process according to the invention wherein
pancreas islet cells are provided under step (d) or (e).

[0333] Pancreas islets are harvested by open biopsy and separated by
conventional enzymatic digestion and Ficol or Hypaqe separation (Page et
al., 2007, Diba. Vas. Dis. Res., 7-12) in a medium enriched with an
autologous platelet concentrate composition according to the invention,
also called RegenPRP®.

[0334] The pancreas islet cell preparation is then injected as a bolus via
the portal vein into the liver.

[0335] The pancreas islet cell preparation according to the invention is
useful for the treatment of type1 diabetes or insulin-dependent diabetes
and for the reversal of hyperglycaemia of diabetes mellitus.

Example 17

Autologous Human Osteoblast Cell Preparation

[0336] Example of autologous cell association according to the invention
can be prepared by using the process according to the invention wherein
osteoblast cells are provided under step (d) or (e).

[0337] Cortical punch bone biopsy is derived from the illiac crest or
equivalent site (maxilla) under local anaesthesia. The bone biopsy is
placed aseptically in DMEM medium at 4° C., or equivalent
transport medium by those experienced in the art of bone and osteoblast
culture ex ViVO. The bone diopsy is then diced and digested in diluted in
10% type-1 collagenase (Sigma or Boehringer) at 37° C. for 15 min
under laminar flow hood. Alternatively, trypsin digestion (Worthington)
may be used alternative. Enzymatic digestion is terminated with three
washes with an autologous platelet concentrate composition according to
the invention, also called RegenPRP® at 10% in DMEM at 4° C.
The preparation is centrifuged, pelleted and resuspended. The bone
fragments are plated on Petri dishes or flasks as explants with
air-lifting technology in an autologous platelet concentrate composition
according to the invention, also called RegenPRP®. The preparation is
cultured at 37° C. with antibiotics, gentimicin and amphotericin-B
under a gas flow of 95% air and 5% CO2. The culture medium is
changed three times per week, each time spiking DMEM medium with 10% vol.
of an autologous platelet concentrate composition according to the
invention. The cell viability and morphology are evaluated three times a
week to assess cell crawling, apoptosis and 3D dimensional monolayer
progression. The formation of microfilament and differentiation is
assessed by inverted microscopy (Olympus®). Absence of bacterial and
viral contamination is checked. Osteoblasts can be engineered onto human
amnion to create cell biocomposite scaffold and cell monolayer
carrier/construct after membrane seeding with 100,000 cells as obtained
above and allowing monolayer membrane expansion over 3-4 weeks allowing
unique construction of osteoblast-amnion-membrane construct for use and
transfer to cover a bone defect or grafted area following non-union of
fracture in any site.

[0338] The osteoblast cell preparation according to the invention is
useful for the treatment of bone defects, bone grafts or bone disorders.